Summary: The structure of the bacterial nucleoid and the forces which maintain its DNA in a highly compact yet accessible form are largely unknown. To approach these problems, we are systematically characterizing nucleoid preparations that are isolated from E. coli under relatively non-denaturing conditions (spermidine nucleoids, Kornberg et al.). In the current work, we use controlled nucleoid denaturation in urea solutions to help delineate some of the stabilizing factors of the nucleoid. We have devised an assay for nucleoid denaturation which can rapidly assess the fraction of DNA unfolding and that is readily applied to large numbers of samples. This "dilatancy" assay uses the pervasive tendency of denatured nucleiods to aggregate under the influence of low centrifugal forces. Nucleoid denaturation occurs in 3 M urea as a cooperative transition. Several properties of this transition indicate a major role for RNA in stabilizing the nucleoids: 1) Pretreatment of the nucleoids with low concentrations of RNase decreases the amount of urea required for half-denaturation (Um value) by 2-fold. 2) Exposure of the cells to rifampicin results in unstable nucleoids. 3) Denaturation is closely matched by RNA degradation. A set of DNA-associated proteins is released from the nucleoid during this transition. Nucleoids isolated from cells having mutations in several of these proteins (Fis, H-NS, StpA) have unchanged Um values. Two new DNA-associated proteins which might be involved in nucleoid function have been identified in E. coli. The amounts of both proteins released from the nucleoids by exogenous DNase are increased 2-fold in a Fis mutant. Partial amino acid sequences of the two proteins are compatible with two previously described "hypothetical proteins" predicted from DNA sequencing studies; one of these has been been shown to be expressed by Ryder et al. and may be an exonuclease. Um is greatly increased in nucleoids from chloramphenicol-treated cells. The components responsible for this "hyperstabilization" are currently under study.